We will continue to study the molecular epidemiology of influenze viruses using biochemical techniques. A study will be undertaken to contrast the evolutionary changes in the earlier H1N1 strains (1950-57) with those observed in the 1950-like H1N1 strains which were reintroduced into the population in 1977. Genetic variation will also be studied with respect to equine 1 influenza viruses (H7N7). These viruses appear to undergo slower antigenic change than human influenza viruses. In addition, we will analyze the genetic composition and antigenic properties of new antigenic "drift" and antigenic "shift" variants of influenza viruses isolated in the future. We have developed a convenient assay to measure amber suppressor tRNA activity in higher eukaryotic cells. This assay is based on the quantitation of the NS1 polypeptide and its Su+ tRNA mediated readthrough product after virus infection. It is planned to extend this method for the measurement of opal and ochre suppressor tRNAs and to search for natural nonsense suppressor tRNA activities in higher eukaryotic cells. With the availability of genetically engineered cells containing Su+ tRNAs it should also be possible to isolate nonsense mutants of influenza virus and to use them for studying the structure/function relationship of influenza viral genes. We also plan to apply the suppressor systems to generate mutant proteins of cloned influenza virus genes which are expressed via SV40 vectors. This approach will allow us to systematically change specific amino acids in a single viral protein in an effort to identify those amino acids which are crucial for the function of the protein.